Next Generation Sequencing Technologies

Transcription

1 Next Generation Sequencing Technologies

2 What is first generation? Sanger Sequencing

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4 DNA Polymerase

5 Base-adding reaction +H +

6 Pros and Cons of Sanger Sequencing Polymerase errors average out Long sequences (~450 bp) Can only do 1 sequence at a time Need a lot of DNA to start with Expensive: 2 /base

7 To solve these cons what do we need? Cheaper Multiplex different samples Smaller starting amount How might you do this? What do you need to be able to do?

8 Design a Sequencer For 1 minute, write down all the things you would need to do to be able to sequence DNA in a multiplexed way. Turn to your neighbors (1-2 people) For 1 minute, discuss the things you would need to do to be able to sequence DNA in a multiplexed way. Be prepared to tell the class what you think you need and why

9 What you need to do multiplexed sequencing Ability to separate individual DNA pieces Ability to observe the sequence of each separated piece individually High sensitivity (as compared to Sanger sequencing)

10 What s different Sequence many sequences at once Technology is paired with DNA sequence agnostic primers Faster than SS Shorter than SS

11 How do we sequence things we don t know the sequence of?

12 Adapt sequences with known sequences This can be done with Sanger too, but need to PCR after this to get enough DNA Mardis, ER; Ann Rev Genom & Hum Gen

13 What you need to do multiplexed sequencing Ability to separate individual DNA pieces Ability to observe the sequence of each separated piece individually High sensitivity (as compared to Sanger sequencing)

14 Emulsion PCR onto beads (454, Ion Torrent)

15 Mardis, E. R. (2013). Next-Generation Sequencing Platforms. Annual Review of Analytical Chemistry, 6(1), doi: /annurevanchem Flow Cell: Bind directly to chip, make bridges (Illumina)

16 What you need to do multiplexed sequencing Ability to separate individual DNA pieces Ability to observe the sequence of each separated piece individually High sensitivity (as compared to Sanger sequencing)

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18 454:2005 Imaging and light based

19 Illumina: 2006 Expose to all 4 bases Add 1 at a time, 3 OH is reversibly blocked Monitor fluorescence Mardis, E. R. (2013). Next-Generation Sequencing Platforms. Annual Review of Analytical Chemistry, 6(1), doi: / annurev-anchem

20 As of 2010, all were imaging based Why might this be problematic? How else might you follow sequencing?

21 Ion Torrent: 2010 On Chips Most accurate ph meter in the world

22 Ion Torrent Expose to single base type at a time Add as many as possible Monitor change in ph

23 Getting DNA onto beads P A P A P P A

24 Getting DNA onto beads P A

25 P Getting DNA onto beads A P P A P A Which strand do we keep when we make this singlestranded for sequencing? A

26 A P Keep sequence that is complementary to the sequence we read from sequencer A 3 5 P P A P A

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29 Actual Sequence on Bead: GTAACTGTCAAACG What happens on Ion Torrent? Cycle through the following bases: T G A C C ATTGACAGTTTGC GTAACTGTCAAACG T G A C TT G A C T G A C T G A C TTT G A C

30 What does the cyclical process mean for our sequencing? On your sheet, figure out how far each of the sequences will get in 5 cycles

31 Histogram of read lengths

32 What is the sequence of the following DNA? TGAC TCTGGTGA

33 Bead with 2 DNAs T T C G G A A ATCTTAGGTA What happens? 2x as many bases as expected

34 Errors Homopolymers AAAAAAA Polymerase adds all at once System becomes saturated How many are there really of a particular base?

35 And Now for Something Completely Different Single Molecule Sequencing

36 Pacific Biosciences: Single Molecule Sequencing (SMRT) Benjamin A Flusberg, Dale R Webster, Jessica H Lee, Kevin J Travers, Eric C Olivares, Tyson A Clark, Jonas Korlach & Stephen W Turner Nature Methods 7, (2010) Published online: 9 May 2010, doi: /nmeth

37 Pacific Biosciences Can get VERY long sequences 5,000-8,000 bases, on average 30,000 bases sometimes 99.99% accurate for each base No averaging, so can find rare SNPs No amplification needed before sequencing, so less bias Differences in rates of addition allow one to measure epigenetic variations Fewer total sequences so generally end up with fewer total bases Much more expensive than the other techniques

38 Oxford Nanopore Technologies: In beta-testing Similar potential benefits as SMRT technology, but without drawbacks of polymerase and use of imaging technologies

39 Oxford Nanopore Technologies

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41 Pros and Cons of NGS Fast Cheap (<1 /Mbase) Lots of data Fewer reads of each base are combined, so less accurate overall Short reads (getting longer, up to ~400 bases now)

42 Activity On table: fill in what you think are the pros and cons of each technology we discussed ~ 2 min Discuss with your neighbors what you each put, generate a consensus list to share with the class ~ 4 min

43 Questions?